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BP1 is a negative modulator of definitive erythropoiesis.

Mpollo MS, Beaudoin M, Berg PE, Beauchemin H, D'Agati V, Trudel M - Nucleic Acids Res. (2006)

Bottom Line: Consistently, the presence of BP1 transgene in fetuses was associated with paleness and lethality.In secondary differentiation, BP1 expression reduced significantly beta-globin gene expression in both primitive and definitive erythroid cells, whereas it impaired only the definitive erythroid cell differentiation.These studies showed that BP1 can negatively modulate adult beta-globin gene expression and definitive erythroid cell differentiation, and suggest that BP1 could play a role in thalassemia.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Development, Institut de Recherches Cliniques de Montreal, Faculte de Medecine de l'Universite de Montreal, 110 ouest avenue des Pins, Montreal, Quebec, Canada H2W 1R7.

ABSTRACT
Beta protein 1 (BP1), a human homeotic transcription factor, is expressed during hematopoeisis in the erythroid lineage. To determine the in vivo role of BP1 in erythropoiesis, we have undertaken two complementary approaches using enforced BP1 expression in both transgenic mice and embryonic stem (ES) cells. Despite repeated attempts, only one adult transgenic BP1 founder mouse among 121 mice was obtained. This mouse presumably survived due to transgene mosaicism because the transgene could not be transmitted. This mouse expressed BP1 and displayed splenomegaly, extramedullary erythropoiesis and severe amyloidosis A in the kidney, a phenotype compatible with thalassemia. Consistently, the presence of BP1 transgene in fetuses was associated with paleness and lethality. In ES cells, BP1 expression in primary differentiation appeared to antagonize adult beta-globin expression. In secondary differentiation, BP1 expression reduced significantly beta-globin gene expression in both primitive and definitive erythroid cells, whereas it impaired only the definitive erythroid cell differentiation. These studies showed that BP1 can negatively modulate adult beta-globin gene expression and definitive erythroid cell differentiation, and suggest that BP1 could play a role in thalassemia.

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Expression analysis of BP1 and β-globin genes in EBs at various stages of primary differentiation. (a) Demonstration of linearity of RT–PCR amplification. Varying quantities of total cDNA aliquots were amplified with primers specific for BP1, β-globin and S16 as internal control to ensure that conditions were within linear range of amplification. The reactions were evaluated semiquantitatively on polyacrylamide gels scanned by phosphoimager and values plotted on linear graphs. (b) Parental ES cells (WT), ES cells containing the empty vector (LCRΔBP1) or ES cells containing BP1 (LCRBP1) were differentiated into embryoid bodies (EBs). Expression analysis of BP1 and of the endogeneous β-globin gene was carried out by RT–PCR on the indicated days of differentiation, using S16 expression as an internal control. Days indicated were selected for analysis of the kinetics of erythroid differentiation since primitive erythroid expression is maximum on day 7 and definitive erythroid lineage expression is high from day 5 to day 12 (11). Semi-quantitative evaluation was carried out for each sample run on the same gel and normalized to the ribosomal RNA control.
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fig4: Expression analysis of BP1 and β-globin genes in EBs at various stages of primary differentiation. (a) Demonstration of linearity of RT–PCR amplification. Varying quantities of total cDNA aliquots were amplified with primers specific for BP1, β-globin and S16 as internal control to ensure that conditions were within linear range of amplification. The reactions were evaluated semiquantitatively on polyacrylamide gels scanned by phosphoimager and values plotted on linear graphs. (b) Parental ES cells (WT), ES cells containing the empty vector (LCRΔBP1) or ES cells containing BP1 (LCRBP1) were differentiated into embryoid bodies (EBs). Expression analysis of BP1 and of the endogeneous β-globin gene was carried out by RT–PCR on the indicated days of differentiation, using S16 expression as an internal control. Days indicated were selected for analysis of the kinetics of erythroid differentiation since primitive erythroid expression is maximum on day 7 and definitive erythroid lineage expression is high from day 5 to day 12 (11). Semi-quantitative evaluation was carried out for each sample run on the same gel and normalized to the ribosomal RNA control.

Mentions: Parental and electroporated ES cells were differentiated in vitro to form EBs (which contain a variety of cell types) and assessed for BP1 expression. The potential of these electroporated ES cells to generate EBs was not significantly affected, as the primary plating efficiency was 17.5 ± 3.9% for parental ES cells, 17.1 ± 4.3% for LCRΔBP1 cells and 14.1 ± 3.5% for LCRBP1 cells (mean ± SD). In addition, LCRBP1 electroporated ES cells yielded similar-sized EBs as the parental and empty vector ES cells (data not shown), suggesting that the number of cells per EB was not altered. During this primary differentiation process, the percentage of hemoglobinized EBs was comparable between the parental and electroporated cells at day 9 (Table 2). To quantify expression levels, we first established the RT–PCR conditions to be within the linear range (Figure 4a). The expression time course of the endogenous adult β-globin gene was similar between wild-type and empty vector control (Figure 4b and data not shown). Noticeably, wild-type ES cells or EBs with empty LCRΔBP1 control did not express BP1 (Figure 4). In two independent experiments, the expression of BP1 in the LCRBP1 clones was highest at days 6 and 9 and decreased from day 12 onwards whereas the β-globin gene showed strongest expression on days 12 and 15. Semi-quantitative analysis of the LCRBP1 clones indicated a ∼1.5- to 2-fold reduction in β-globin expression normalized to S16 on days 12 and 15 relative to empty vector control. Consistently, quantification by real-time PCR of EBs at day 9 showed a decrease in β-globin expression levels by at least 4-fold, a time when BP1 is strongest (Table 3 and Figure 4). Hence the LCRBP1 clones indicated that BP1 might antagonize expression of the murine β-globin gene. Conversely, repression of BP1 in the erythroleukemic K562 cell line was shown to enhance endogeneous human β-globin gene expression (3).


BP1 is a negative modulator of definitive erythropoiesis.

Mpollo MS, Beaudoin M, Berg PE, Beauchemin H, D'Agati V, Trudel M - Nucleic Acids Res. (2006)

Expression analysis of BP1 and β-globin genes in EBs at various stages of primary differentiation. (a) Demonstration of linearity of RT–PCR amplification. Varying quantities of total cDNA aliquots were amplified with primers specific for BP1, β-globin and S16 as internal control to ensure that conditions were within linear range of amplification. The reactions were evaluated semiquantitatively on polyacrylamide gels scanned by phosphoimager and values plotted on linear graphs. (b) Parental ES cells (WT), ES cells containing the empty vector (LCRΔBP1) or ES cells containing BP1 (LCRBP1) were differentiated into embryoid bodies (EBs). Expression analysis of BP1 and of the endogeneous β-globin gene was carried out by RT–PCR on the indicated days of differentiation, using S16 expression as an internal control. Days indicated were selected for analysis of the kinetics of erythroid differentiation since primitive erythroid expression is maximum on day 7 and definitive erythroid lineage expression is high from day 5 to day 12 (11). Semi-quantitative evaluation was carried out for each sample run on the same gel and normalized to the ribosomal RNA control.
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Related In: Results  -  Collection

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fig4: Expression analysis of BP1 and β-globin genes in EBs at various stages of primary differentiation. (a) Demonstration of linearity of RT–PCR amplification. Varying quantities of total cDNA aliquots were amplified with primers specific for BP1, β-globin and S16 as internal control to ensure that conditions were within linear range of amplification. The reactions were evaluated semiquantitatively on polyacrylamide gels scanned by phosphoimager and values plotted on linear graphs. (b) Parental ES cells (WT), ES cells containing the empty vector (LCRΔBP1) or ES cells containing BP1 (LCRBP1) were differentiated into embryoid bodies (EBs). Expression analysis of BP1 and of the endogeneous β-globin gene was carried out by RT–PCR on the indicated days of differentiation, using S16 expression as an internal control. Days indicated were selected for analysis of the kinetics of erythroid differentiation since primitive erythroid expression is maximum on day 7 and definitive erythroid lineage expression is high from day 5 to day 12 (11). Semi-quantitative evaluation was carried out for each sample run on the same gel and normalized to the ribosomal RNA control.
Mentions: Parental and electroporated ES cells were differentiated in vitro to form EBs (which contain a variety of cell types) and assessed for BP1 expression. The potential of these electroporated ES cells to generate EBs was not significantly affected, as the primary plating efficiency was 17.5 ± 3.9% for parental ES cells, 17.1 ± 4.3% for LCRΔBP1 cells and 14.1 ± 3.5% for LCRBP1 cells (mean ± SD). In addition, LCRBP1 electroporated ES cells yielded similar-sized EBs as the parental and empty vector ES cells (data not shown), suggesting that the number of cells per EB was not altered. During this primary differentiation process, the percentage of hemoglobinized EBs was comparable between the parental and electroporated cells at day 9 (Table 2). To quantify expression levels, we first established the RT–PCR conditions to be within the linear range (Figure 4a). The expression time course of the endogenous adult β-globin gene was similar between wild-type and empty vector control (Figure 4b and data not shown). Noticeably, wild-type ES cells or EBs with empty LCRΔBP1 control did not express BP1 (Figure 4). In two independent experiments, the expression of BP1 in the LCRBP1 clones was highest at days 6 and 9 and decreased from day 12 onwards whereas the β-globin gene showed strongest expression on days 12 and 15. Semi-quantitative analysis of the LCRBP1 clones indicated a ∼1.5- to 2-fold reduction in β-globin expression normalized to S16 on days 12 and 15 relative to empty vector control. Consistently, quantification by real-time PCR of EBs at day 9 showed a decrease in β-globin expression levels by at least 4-fold, a time when BP1 is strongest (Table 3 and Figure 4). Hence the LCRBP1 clones indicated that BP1 might antagonize expression of the murine β-globin gene. Conversely, repression of BP1 in the erythroleukemic K562 cell line was shown to enhance endogeneous human β-globin gene expression (3).

Bottom Line: Consistently, the presence of BP1 transgene in fetuses was associated with paleness and lethality.In secondary differentiation, BP1 expression reduced significantly beta-globin gene expression in both primitive and definitive erythroid cells, whereas it impaired only the definitive erythroid cell differentiation.These studies showed that BP1 can negatively modulate adult beta-globin gene expression and definitive erythroid cell differentiation, and suggest that BP1 could play a role in thalassemia.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Development, Institut de Recherches Cliniques de Montreal, Faculte de Medecine de l'Universite de Montreal, 110 ouest avenue des Pins, Montreal, Quebec, Canada H2W 1R7.

ABSTRACT
Beta protein 1 (BP1), a human homeotic transcription factor, is expressed during hematopoeisis in the erythroid lineage. To determine the in vivo role of BP1 in erythropoiesis, we have undertaken two complementary approaches using enforced BP1 expression in both transgenic mice and embryonic stem (ES) cells. Despite repeated attempts, only one adult transgenic BP1 founder mouse among 121 mice was obtained. This mouse presumably survived due to transgene mosaicism because the transgene could not be transmitted. This mouse expressed BP1 and displayed splenomegaly, extramedullary erythropoiesis and severe amyloidosis A in the kidney, a phenotype compatible with thalassemia. Consistently, the presence of BP1 transgene in fetuses was associated with paleness and lethality. In ES cells, BP1 expression in primary differentiation appeared to antagonize adult beta-globin expression. In secondary differentiation, BP1 expression reduced significantly beta-globin gene expression in both primitive and definitive erythroid cells, whereas it impaired only the definitive erythroid cell differentiation. These studies showed that BP1 can negatively modulate adult beta-globin gene expression and definitive erythroid cell differentiation, and suggest that BP1 could play a role in thalassemia.

Show MeSH
Related in: MedlinePlus